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Transforming Ability of MEN2A-RET Requires Activation of the Phosphatidylinositol 3-Kinase/AKT Signaling Pathway*

  • Carine Segouffin-Cariou
    Affiliations
    Laboratoire de Génétique, CNRS UMR 5641, Domaine Rockefeller, 8 Avenue Rockefeller, 69373 Lyon, Cedex 08, France
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  • Marc Billaud
    Correspondence
    To whom correspondence should be addressed. Tel.: 33 478 77 72 13; Fax: 33 478 77 72 20
    Affiliations
    Laboratoire de Génétique, CNRS UMR 5641, Domaine Rockefeller, 8 Avenue Rockefeller, 69373 Lyon, Cedex 08, France
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  • Author Footnotes
    * This work was supported by the Ligue Nationale Contre le Cancer and by the EC Project BMH4-97-2157.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:February 04, 2000DOI:https://doi.org/10.1074/jbc.275.5.3568
      The RET gene codes for a receptor tyrosine kinase that plays a crucial role during the development of both the enteric nervous system and the kidney. Germ line missense mutations at one of six codons specifying extracytoplasmic cysteines are responsible for two related cancer disorders as follows: multiple endocrine neoplasia type2A (MEN2A) and familial medullary thyroid carcinoma (FMTC). MEN2A and FMTC mutations result in a constitutive catalytic activity and as a consequence convert RET into a dominantly acting transforming gene. Although it has been shown that RET-MEN2 mutants activate several transduction pathways, their respective contribution to the neoplastic phenotype remains poorly understood. Over the past few years, it has become increasingly clear that the transforming ability of several viral and cellular oncoproteins depends on their capacity to activate phosphatidylinositol 3-kinase (PI3K). We now report that RET carrying a representative MEN2A mutation at Cys-634 (termed RET-MEN2A) activates PI3K and its downstream effector, the serine/threonine kinase AKT/protein kinase B. Previous studies have demonstrated that mutation of Tyr-1062, which is the intracellular docking site for Shc and Enigma on RET, abolishes the RET-MEN2A transforming activity. We provide evidence that mutation of Tyr-1062 abrogates the binding of the p85 regulatory subunit of PI3K to RET-MEN2A and the subsequent stimulation of the PI3K/AKT pathway. Furthermore, infection of rat fibroblasts with a retrovirus expressing a dominant-interfering form of PI3K suppresses RET-MEN2A-dependent transformation, whereas overexpression of AKT enhances the RET-MEN2A oncogenic potential. In summary, these data are consistent with the notion that RET-mediated cell-transforming effect is critically dependent on the activation of the PI3K/AKT pathway.
      GDNF
      glial cell line-derived neurotrophic factor
      PI3K
      phosphatidylinositol 3-kinase
      MEN2
      multiple endocrine neoplasia type 2
      FMTC
      familial medullary thyroid carcinoma
      MTC
      medullary thyroid carcinomal
      PtdIns
      phosphatidylinositol
      HA
      hemagglutinin
      wt
      wild type
      GST
      glutathione S-transferase
      The RET gene codes for a transmembrane tyrosine kinase that displays a cadherin-like domain and a cysteine-rich motif in its extracellular region (
      • Takahashi M.
      • Buma Y.
      • Iwamoto T.
      • Inaguma Y.
      • Ideka H.
      • Hiai H.
      ). RET is a component of a multiprotein complex that acts as a receptor for four closely related molecules, glial cell line-derived neurotrophic factor (GDNF),1 neurturin, artemin, and persephin (see Ref.
      • Airaskinen M.S.
      • Titievsky A.
      • Saarma M.
      for review). In addition to RET, this complex contains glycosylphosphatidylinositol-anchored receptors called GFRαs that directly interact with the different ligands and induce homodimerization of RET, thereby leading to the activation of the tyrosine kinase (
      • Airaskinen M.S.
      • Titievsky A.
      • Saarma M.
      ). To date, four homologous GFRαs have been identified, and the ligand specificity of the multisubunit receptor is dictated by the nature of the glycosylphosphatidylinositol-bound protein since GFRα-1, -2, -3, and -4 exhibit a higher affinity for GDNF, neurturin, artemin, and persephin, respectively (
      • Airaskinen M.S.
      • Titievsky A.
      • Saarma M.
      ). Gene knock out studies have revealed that null mice lacking either RET,GFRα-1, or GDNF die soon after birth and share strikingly analogous phenotypes including renal agenesis and absence of the enteric nervous system in the intestine (
      • Airaskinen M.S.
      • Titievsky A.
      • Saarma M.
      ). Evidence has been further provided that GDNF is both a neurotrophic and a urotrophic factor capable of both protecting enteric neurons from apoptotic cell death and promoting growth and branching of the ureteric bud, respectively (
      • Airaskinen M.S.
      • Titievsky A.
      • Saarma M.
      ).
      Germ line mutations of RET are responsible for multiple endocrine neoplasia type 2 (MEN2), a dominantly inherited cancer syndrome that encompasses three related clinical subtypes, each typified by the involvement of a specific spectrum of endocrine tissues involved. MEN type 2A (MEN2A) is characterized by the association of medullary thyroid carcinoma (MTC), a malignant tumor arising from calcitonin-secreting C-cell, pheochromocytoma, and more occasionally parathyroid hyperplasia or adenoma. MEN type 2B (MEN2B) is a more rare and a more severe syndrome defined by the presence of MTC, pheochromocytoma, mucosal neuromas, hyperplasia of the enteric nervous plexuses, and skeletal abnormalities. Finally, in the familial form of MTC (FMTC), thyroid cancer is the sole clinical manifestation (see Ref.
      • Ponder B.A.
      for review). Missense mutations at one of six cysteines codons (Cys-609, -611, -618, -620, -630, and -634), which result in the substitution of any one of these extracytoplasmic cysteines by a different amino acid, are responsible for the majority of MEN2A and a large fraction of FMTC cases (
      • Ponder B.A.
      ,
      • Donis-Keller H.
      • Dou S.
      • Chi D.
      • Carlson K.
      • Toshima K.
      • Lairmore T.
      • Howe J.
      • Moley J.
      • Goodfellow P.
      • Wells S.J.
      ,
      • Mulligan L.
      • Kwok J.
      • Healey C.
      • Elsdon M.
      • Eng C.
      • Gardner E.
      • Love D.
      • Mole S.
      • Moore J.
      • Papi L.
      • Ponder M.A.
      • Telenius H.
      • Tunnacliffe A.
      • Ponder B.A.J.
      ,
      • Eng C.
      • Clayton D.
      • Schuffenecker I.
      • Lenoir G.
      • Cote G.
      • Gagel R.
      • Ploos V.A.
      • Lips C.
      • Nishisho I.
      • Takai S.-I.
      • Marsh D.
      • Robinson B.
      • Frank-Raue K.
      • Raue F.
      • Xue F.
      • Noll W.
      • Romei C.
      • Pacini F.
      • Fink M.
      • Niederle B.
      • Zedenius J.
      • Nordenskjold M.
      • Komminoth P.
      • Hendy G.
      • Gharib H.
      • Thibodeau S.
      • Lacroix A.
      • Frilling A.
      • Ponder B.
      • Mulligan L.
      ). Moreover, FMTC and rare cases of MEN2A have been also ascribed to five mutations singly affecting amino acids within the tyrosine kinase domain (
      • Ponder B.A.
      ). MEN2B is exclusively caused by two distinct mutations involving residues located within the kinase catalytic domain and resulting in the replacement of either Ala-883 for a Phe or Met-918 for a Thr, the latter mutation being found the most frequently (
      • Ponder B.A.
      ).
      All MEN2 mutations tested so far have been shown to induce a ligand-independent constitutive activation of RET (
      • Asai N.
      • Iwashita T.
      • Matsuyama M.
      • Takahashi M.
      ,
      • Santoro M.
      • Carlomagno F.
      • Romano A.
      • Bottaro D.
      • Dathan N.
      • Grieco M.
      • Fusco A.
      • Vecchio G.
      • Matoskova B.
      • Kraus M.
      • Di Fiore P.
      ). Cysteine mutations result in the aberrant RET homodimerization via the formation of inter-chain disulfide bridges (
      • Asai N.
      • Iwashita T.
      • Matsuyama M.
      • Takahashi M.
      ,
      • Santoro M.
      • Carlomagno F.
      • Romano A.
      • Bottaro D.
      • Dathan N.
      • Grieco M.
      • Fusco A.
      • Vecchio G.
      • Matoskova B.
      • Kraus M.
      • Di Fiore P.
      ,
      • Iwashita T.
      • Murakami H.
      • Asai N.
      • Takahashi M.
      ,
      • Chappuis-Flament S.
      • Pasini A.
      • De Vita G.
      • Segouffin-Cariou C.
      • Fusco A.
      • Attie T.
      • Lenoir G.
      • Santoro M.
      • Billaud M.
      ), whereas MEN2B mutations are thought to alter the substrate specificity of the tyrosine kinase (
      • Santoro M.
      • Carlomagno F.
      • Romano A.
      • Bottaro D.
      • Dathan N.
      • Grieco M.
      • Fusco A.
      • Vecchio G.
      • Matoskova B.
      • Kraus M.
      • Di Fiore P.
      ,
      • Songyang Z.
      • Carraway K.L.
      • Eck M.J.
      • Harrison S.C.
      • Feldman R.A.
      • Mohammadi M.
      • Schlessinger J.
      • Hubbard S.R.
      • Smith D.P.
      • Eng C.
      • Lorenzo M.J.
      • Ponder B.A.J.
      • Mayer B.J.
      • Cantley L.C.
      ,
      • Bocciardi R.
      • Mograbi B.
      • Pasini B.
      • Borrello M.G.
      • Pierotti M.A.
      • Bourget I.
      • Fisher S.
      • Romeo G.
      • Rossi B.
      ). In addition, transgenic mice expressing an MEN2A/FMTC allele in the thyroid C-cell developed C-cell hyperplasia and MTC (
      • Michiels F.M.
      • Chappuis S.
      • Caillou B.
      • Pasini A.
      • Talbot M.
      • Monier R.
      • Lenoir G.M.
      • Feunteun J.
      • Billaud M.
      ), whereas mice expressing MEN2B allele under the control of the dopamine β-hydroxylase promoter displayed ganglioneuromas and renal malformations (
      • Sweetser D.A.
      • Froelick G.J.
      • Matsumoto A.M.
      • Kafer K.E.
      • Marck B.
      • Palmiter R.D.
      • Kapur R.P.
      ). Taken together, these results demonstrate thatMEN2 mutations transform RET into a dominantly acting oncogene and suggest that the uncontrolled stimulation of signaling pathways downstream of RET accounts for its transforming ability. Upon RET activation, the autophosphorylated tyrosine residues Tyr-905, Tyr-1015, Tyr-1062, and Tyr-1096 act as docking sites for the transduction effectors Grb7/Grb10/Grb14, phospholipase Cγ, Shc/Enigma, and Grb2, respectively (
      • Santoro M.
      • Wong W.T.
      • Aroca P.
      • Santos E.
      • Matoskova B.
      • Grieco M.
      • Fusco A.
      • Di Fiore P.P.
      ,
      • Asai N.
      • Murakami H.
      • Iwashita T.
      • Takahashi M.
      ,
      • Borrello M.G.
      • Alberti L.
      • Arighi E.
      • Bongarzone I.
      • Battistini C.
      • Bardelli A.
      • Pasini B.
      • Piutti C.
      • Rizzetti M.G.
      • Mondellini P.
      • Radice M.T.
      • Pierotti M.
      ,
      • Durick K.
      • Wu R.-Y.
      • Gill G.N.
      • Taylor S.S.
      ,
      • Liu X.
      • Vega Q.C.
      • Deckers R.A.
      • Pandey A.
      • Worby C.A.
      • Dixon J.E.
      ,
      • Pandey A.
      • Liu X.
      • Dixon J.E.
      • Di Fiore P.P.
      • Dixit V.M.
      ,
      • Arighi E.
      • Alberti L.
      • Torriti F.
      • Ghizzoni S.
      • Rizetti M.G.
      • Pelicci G.
      • Pasini B.
      • Bongarzone I.
      • Piutti C.
      • Pierotti M.A.
      • Borrello M.G.
      ,
      • Lorenzo M.J.
      • Gish G.D.
      • Houghton C.
      • Stonehouse T.J.
      • Pawson T.
      • Ponder B.A.J.
      • Smith D.P.
      ,
      • Alberti L.
      • Borello M.G.
      • Ghizzoni S.
      • Torriti F.
      • Rizzetti M.G.
      • Pierotti M.A.
      ). The Tyr-1062 residue is an essential site since its mutation impairs the oncogenic potential of all MEN2 forms of RET tested so far (
      • Asai N.
      • Murakami H.
      • Iwashita T.
      • Takahashi M.
      ,
      • Iwashita T.
      • Kato M.
      • Murakami H.
      • Asai N.
      • Ishiguro Y.
      • Ito S.
      • Iwata Y.
      • Kawai K.
      • Asai M.
      • Kurokawa K.
      • Kajita H.
      • Takahashi M.
      ). Furthermore, it is known that RET-MEN2 relays an intracellular signal by activating several signaling cascades including the Ras/mitogen-activated protein kinase and the Jun and Src kinases (
      • van Weering D.
      • Medema J.P.
      • van Puijenbroek A.
      • Burgering B.
      • Baas P.
      • Bos J.
      ,
      • Marshall G.
      • Peaston A.
      • Hocker J.
      • Smith S.
      • Hansford L.
      • Tobias V.
      • Norris M.
      • Haber M.
      • Smith D.
      • Lorenzo M.
      • Ponder B.
      • Hancock J.
      ,
      • van Puijenbroek A.A.
      • van Weering D.H.
      • van den Brink C.E.
      • Bos J.L.
      • van der Saag P.T.
      • de Laat S.W.
      • den Hertog J.
      ,
      • Chiariello M.
      • Visconti R.
      • Carlomagno F.
      • Medillo R.M.
      • Bucci C.
      • de Franciscis V.
      • Fox G.
      • Jing S.
      • Coso O.
      • Gutkind J.S.
      • Fusco A.
      • Santoro M.
      ,
      • Melillo R.
      • Barone M.
      • Lupoli G.
      • Cirafici A.M.
      • Carlomagno F.
      • Visconti R.
      • Matoskova B.
      • Di Fiore P.P.
      • Vecchio G.
      • Fusco A.
      • Santoro M.
      ). However, evidence that the sustained stimulation of these respective pathways contributes to the RET-MEN2 transforming capacity is still limited.
      Phosphoinositide 3-kinases (PI3K) are crucial transducers that control multiple biologic responses, such as mitogenesis, protection from programmed cell death, and cellular motility (reviewed in Refs.
      • Marte B.
      • Downward J.
      and
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      ). Class I of PI3K is a heterodimeric enzyme comprising a regulatory subunit of 85 kDa, p85, and a catalytic component of 110 kDa, p110. The p85 subunit contains two SH2 domains that can interact with phosphotyrosine. PI3K binds activated receptor tyrosine kinases either directly via the interaction of p85 to phosphorylated tyrosines or indirectly through the association of p85 with other transduction effectors possessing tyrosine docking sites, such as IRS-1, Gab-1, or c-Cbl (
      • Marte B.
      • Downward J.
      ,
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      ). The translocation of PI3K to the inner face of the plasma membrane locates the enzyme closer to upstream regulators (such as Ras) and recruits PI3K in proximity to its lipid substrates. Once activated, class I PI3Ks catalyze the phosphorylation of phosphoinositide on position 3 of the inositol ring and generate PtdIns(3,4)P2 and PtdIns(3,4,5)P3. These lipid products act as binding sites for transduction effectors, and this interaction regulates their subcellular localization and modulates their activity. The serine/threonine kinase protein kinase B/AKT has been identified as a crucial downstream effector that mediates the effects of PI3K on cell survival, cell proliferation, and on the response to insulin (
      • Coffer P.
      • Jin J.
      • Woodgett J.
      ,
      • Khwaja A.
      ).
      We now report that Rat1 cell line expressing an MEN2A form of RET displays a constitutive PI3K activity. Furthermore, mutation of Tyr-1062 blocked the RET-mediated activation of PI3K, and expression of a dominant-interfering mutant form of PI3K drastically reduced the transforming ability of RET-MEN2A.

      DISCUSSION

      Several recent studies have revealed that dysregulation of the PI3K/AKT pathway is a frequent occurrence during tumorigenesis (reviewed in Refs.
      • Marte B.
      • Downward J.
      ,
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      , and
      • Cantley L.
      • Neel B.
      ). In this study, we found that PI3K and AKT were constitutively activated in rat fibroblasts expressing a MEN2A mutant form of RET. We further show that interference with the signal transduced through PI3K inhibited the RET-MEN2A transforming capacity measured by the ability of cells to grow without anchorage.

      Mechanisms of PI3K Activation by RET-MEN2A

      The recruitment of p85 to activated tyrosine kinase receptors is a crucial event for the activation of PI3K (reviewed in Ref.
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      ). With regard to this mechanism, we showed that RET-MEN2A bound in vitro to a recombinant GST protein fused to the amino-terminal SH2 domain of p85 (GST-p85) and also, although to a lesser extent, to the carboxyl-terminal SH2 domain of p85 (data not shown). Furthermore, p85 was found either phosphorylated on tyrosine residues or associated in a multimolecular complex with proteins containing phosphotyrosine in cells expressing RET-MEN2A (data not shown). These results indicate that p85 might either directly recognize a phosphotyrosine docking site on RET or indirectly contact RET through its association with bridging signaling molecules. Our results support the latter hypothesis as follows: (i) mutation of Tyr-1062 in RET-MEN2A prevents the binding of p85 and considerably reduces the activation of the PI3K/AKT pathway; (ii) mutation of Tyr-981, which is the RET tyrosine residue surrounded by the peptidic sequence the closest to the optimal p85-binding site, does not significantly alter either the RET-MEN2A-p85 interaction or the activation of AKT. It is known that Shc and Enigma interact with Tyr-1062 (
      • Asai N.
      • Murakami H.
      • Iwashita T.
      • Takahashi M.
      ,
      • Durick K.
      • Wu R.-Y.
      • Gill G.N.
      • Taylor S.S.
      ,
      • Arighi E.
      • Alberti L.
      • Torriti F.
      • Ghizzoni S.
      • Rizetti M.G.
      • Pelicci G.
      • Pasini B.
      • Bongarzone I.
      • Piutti C.
      • Pierotti M.A.
      • Borrello M.G.
      ,
      • Lorenzo M.J.
      • Gish G.D.
      • Houghton C.
      • Stonehouse T.J.
      • Pawson T.
      • Ponder B.A.J.
      • Smith D.P.
      ), and therefore it is possible that either one of these two proteins recruit p85. Alternatively, additional adaptor proteins, yet to be characterized, might recognize Tyr-1062 and associate with p85. Interestingly, Murakami and co-workers (
      • Murakami H.
      • Iwashita T.
      • Asai N.
      • Shimono Y.
      • Iwata Y.
      • Kawai K.
      • Takahashi M.
      ) have recently shown that the Grb2-associated binder-1 protein (Gab-1) is phosphorylated on tyrosine in cells expressing RET-MEN2. Gab-1 contains several docking sites for p85 and is known to mediate PI3K activation (
      • Holgado-Madruga M.
      • Emlet D.R.
      • Moscatello D.K.
      • Godwin A.K.
      • Wong A.J.
      ,
      • Holgado-Madruga M.
      • Moscatello D.
      • Emlet D.
      • Dieterich R.
      • Wong A.
      ), thus raising the possibility that Shc connects RET to PI3K via Grb2 and Gab-1. Although we cannot formally exclude that RET is also able to tether p85 directly, our data indicate that Tyr-981 is not a genuine p85-binding site. These findings are corroborated by the demonstration that mutation of Tyr-981 does not impair the transforming activity of RET-MEN2A and RET-MEN2B (
      • Asai N.
      • Murakami H.
      • Iwashita T.
      • Takahashi M.
      ).
      It is now well established that activated Ras interacts with p110 and contributes to the activation of PI3K (reviewed in Refs.
      • Marte B.
      • Downward J.
      and
      • Downward J.
      ). Accordingly, the failure of RET-MEN2A Y1062F mutant to trigger the PI3K pathway might also reflect its inability to activate Ras via the formation of the Shc-Grb2-SOS complex. This idea is sustained by a number of recent experiments that showed that the expression of a dominant-interfering form of Ras (Asn-17 Ras) partially inhibits RET/GDNF-mediated induction of AKT (
      • van Weering D.
      • de Rooij J.
      • Marte B.
      • Downward J.
      • Bos J.
      • Burgering B.
      ). It is likely that both the recruitment of p85/p110 in the vicinity of RET and the activation of Ras synergize to stimulate PI3K. In this case, Tyr-1062 would be a crucial site for both molecular mechanisms that converge on the activation of PI3K. A similar situation has been documented for the TrkA receptor tyrosine kinase that possesses an Shc-docking site and whose role is essential for the activation of Ras and PI3K (
      • Hallberg B.
      • Ashcroft M.
      • Loeb D.
      • Kaplan D.
      • Downward J.
      ).

      Activation of AKT Is Required for RET-MEN2A Transforming Activity

      It is known that PI3K transduces intracellular signals through multiple effector pathways that contribute to a plethora of biological responses (
      • Marte B.
      • Downward J.
      ,
      • Fruman D.A.
      • Meyers R.E.
      • Cantley L.C.
      ). The protein kinase AKT is a crucial downstream target that delivers mitogenic and anti-apoptotic signals (
      • Coffer P.
      • Jin J.
      • Woodgett J.
      ,
      • Khwaja A.
      ). Consistent with these data, AKT was found to be activated in cells expressing RET-MEN2A, and this activation was PI3K-dependent. Furthermore, overexpression of AKT markedly enhanced the transforming effect of RET-MEN2A, thus indicating that AKT is a limiting effector for RET-MEN2A oncogenic potential. In addition, expression of dominant-negative forms of AKT was recently found to inhibit RET-MEN2A oncogenic activity (data not shown).
      C. Segouffin-Cariou and M. Billaud, unpublished data.
      Previous studies have revealed that the mechanisms by which AKT prevents cell death require its ability to phosphorylate and subsequently inactivate pro-apoptotic factors such as BAD (
      • Datta S.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.
      ,
      • del Peso L.
      • Gonzalez-Garcia M.
      • Page C.
      • Herrera R.
      • Nunez G.
      ), caspase 9 (
      • Cardone M.H.
      • Roy N.
      • Stennicke H.R.
      • Salvesen G.S.
      • Franke T.F.
      • Stanbridge E.
      • Frisch S.
      • Reed J.C.
      ), and transcriptions factors of the forkhead family homologous to FKHR (
      • Brunet A.
      • Bonni A.
      • Zigmond M.
      • Lin M.
      • Juo P.
      • Hu L.
      • Anderson M.
      • Arden K.
      • Blenis J.
      • Greenberg M.
      ,
      • Kops G.
      • de Ruiter N.
      • De Vries-Smits A.
      • Powell D.
      • Bos J.
      • Burgering B.
      ). In accordance with these findings, we obtained evidence that FKHRL1, one member of the FKHR family characterized as a substrate of AKT, is phosphorylated in cells transformed by RET-MEN2A and thus excluded from the nucleus (data not shown).2 This exclusion is likely to be due to the sequestration of the phosphorylated form of FKHRL1 by 14-3-3 proteins, as demonstrated by Brunet and co-workers (
      • Brunet A.
      • Bonni A.
      • Zigmond M.
      • Lin M.
      • Juo P.
      • Hu L.
      • Anderson M.
      • Arden K.
      • Blenis J.
      • Greenberg M.
      ) in a different experimental context. Furthermore, AKT phosphorylates and suppresses the catalytic activity of the glycogen synthase kinase 3α (GSK-3 α), an enzyme involved not only in the regulation of glucose homeostasis but also implicated in the regulation of both apoptosis and mitogenesis via its control of β-catenin and cyclin D1 turnover (Ref.
      • Diehl J.
      • Cheng M.
      • Roussel M.
      • Sherr C.
      and references therein). Finally, several recent reports have shown that AKT increases the activity of endothelial nitric-oxide synthase and NF-kB thus leading to the production of nitric oxide and to the transcription of genes coding for anti-apoptotic proteins (
      • Fulton D.
      • Gratton J.P.
      • McCabe T.J.
      • Fontana J.
      • Fujio Y.
      • Walsh K.
      • Franke T.F.
      • Papapetropoulos A.
      • Sessa W.C.
      ,
      • Dimmeler S.
      • Fleming I.
      • Fisslthaler B.
      • Herman C.
      • Busse R.
      • Zeilher A.M.
      ,
      • Ozes O.
      • Mayo L.
      • Gustin J.
      • Pfeffer S.
      • Pfeffer L.
      • Donner D.
      ,
      • Romashkova J.
      • Makarov S.
      ). Future experiments that aim at analyzing the respective roles of these downstream targets of AKT should enable us to elucidate the molecular mechanisms presumably involved in the etiology of MEN2 tumors.

      Role of the Dysregulation of PI3K during RET-MEN2-mediated Transformation

      Expression of Δp85 was found sufficient to reduce the oncogenic ability of RET-MEN2A, thus raising a question concerning the biological role of PI3K/AKT signaling during cellular transformation. The signal transmitted via PI3K/AKT is known to promote cell survival and to control the G1 to S phase transition of the cell cycle in response to different growth factors (
      • Klippel A.
      • Escobedo M.-A.
      • Wachowicz M.
      • Apell G.
      • Brown T.
      • Giedlin M.
      • Kavanaugh M.
      • Williams L.
      ). However, in our system it is unlikely that RET-MEN2A mediated its transforming effect by blocking cell death, since fibroblasts expressing RET wild type or RET-MEN2A were cultured in the presence of serum and did not display apoptotic features. It appears more likely that protracted activation of PI3K via RET-MEN2A results in cellular changes that characterize tumor growth, such as anchorage-independent growth. This is consistent with a study that reported that chronic stimulation of PI3K in combination with serum causes cellular modifications specific to transformed cells, whereas activation of PI3K in the absence of serum leads to apoptosis (
      • Klippel A.
      • Escobedo M.-A.
      • Wachowicz M.
      • Apell G.
      • Brown T.
      • Giedlin M.
      • Kavanaugh M.
      • Williams L.
      ). Yet, it remains possible that in vivo both the PI3K-mediated protection from programmed cell death and the mitogenic effect are required in RET-MEN2 oncogenic activity, a question we are currently exploring. It has been recently demonstrated that the platelet-derived growth factor induces two waves of PI3K activity, the second wave being required for platelet-derived growth factor-dependent DNA synthesis (
      • Jones S.
      • Klinghoffer R.
      • Prestwich G.
      • Toker A.
      • Kazlauskas A.
      ). Therefore, it is plausible that aberrant accumulation throughout the cell cycle of PI3K-derived lipid products may account for the capacity of RET-MEN2 to promote cell cycle abnormalities and anchorage-independent growth. While this work was being completed, Murakami and co-workers (
      • Murakami H.
      • Iwashita T.
      • Asai N.
      • Shimono Y.
      • Iwata Y.
      • Kawai K.
      • Takahashi M.
      ) reported that RET-MEN2B is a more potent activator of PI3K than RET-MEN2A, thus suggesting that high levels of activity of this lipid kinase might contribute to the pathogenesis of clinical features specific to MEN2B.
      In conclusion, these results provide evidence that the prolonged activation of the PI3K/AKT signaling pathway is a key event that accounts for the oncogenic ability of the MEN2A form of RET. It now becomes essential to investigate whether mutant forms of RET chronically activate the PI3K pathway in endocrine cells that are affected in MEN2 and to determine if this activation is required for the development of tumors.

      Acknowledgments

      We thank Anne Brunet for the kind gift of anti-FKHRL1 antibodies, Julian Downward for the vector expressing Δp85, and Janet Hall for critical reading of the manuscript.

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